It was discovered in the late 1920s that certain “essential” dietary fatty acids must be present in effective quantities for normal growth and health in rats to ensue (Burr & Burr J. Biol. Chem., 82: 345-367 1929). Epidemiological data collected from human populations beginning in the 1940s then suggested relatively high dietary intakes of n-3 HUFA may be protective against the development of a number of medical conditions and that low n-3 intake may increase risk (Sinclair. Lancet 1:381-3 1956; Bang et al., Lancet 1:1143-5.1971; Hirai of al., Lancet 2:1132-3. 1980; Kromhout of al Am J. Clin. Nutr., 85:1142-1147).
In recent decades supplementation studies incorporating individual omega-3 highly unsaturated fatty acids (n-3 HUFA) in the diet of humans have demonstrated beneficial health effects of individual dietary n-3 HUFA. In particular, human dietary supplementation studies incorporating relatively pure forms of the n-3 HUFA eicosapentaenoic acid (EPA) have suggested this nutrient may promote health and ameliorate or even reverse the effects of a range of common diseases, including but not limited to certain forms of cardiovascular disease and depression (Yokoyama et al., Lancet 369:1062-1063. 2007; Peet & Horrobin Arch. Gen. Psych. 59(10) 913-9 2002).
The therapeutic effect of dietary supplementation with concentrated forms of EPA are dependent to some extent on purity. High purity dose forms have an advantage in terms of increased bioavailability. Furthermore the desired effects of EPA are limited or even reversed by the co-consumption of undesired molecules; (as herein defined) in particular docosohexaenoic acid (DHA); also AA and other omega-3 and 6 fatty acids in general. Therefore to enable effective pharmaceutical or therapeutic use of EPA, high purity dose forms, free of the undesired molecules, are required.
Should the demand for high purity EPA increase, which seems likely, large numbers of clinically or subclinically diseased persons may come to depend on continuity of supply long term to maintain quality of life. To date, however, commercial manufacturers have not been capable of economically producing EPA-only compositions with relatively high EPA purities which are at the same time devoid of undesired molecules.
Reasons include: (1) The raw material for commercial production is exclusively limited to particular fish oils containing high levels of undesired molecules. (2) The undesired molecules contained in fish oil are structurally or physico-chemically similar to EPA and cannot be easily removed during purification (3) The cost of further purification rises in a non-linear fashion with increasing purity.
Consequently even at EPA purities up to around the high 90th percentile up to 1% or more of these undesired molecules may remain.
Purification processes are also rendered less efficient by the relatively complex mixture of fatty acids, and a high degree of natural variability contained in fish oil.
The practical effect of the abovementioned factors is that commercial products currently available that contain high purity EPA may also contain unacceptably high concentrations of the abovementioned undesired molecules for therapeutic use. Furthermore the high cost of purifying fish oil to an extent where only small amounts of undesired molecules remain constrain the use of these ultrapure compositions.
Up to 15 kgs of high EPA fish oil are required to produce 1 kg of highly purified EPA in current purification processes. Because the efficiency of such manufacturers is sensitive to the initial concentration of EPA these are based on fish caught with a high percentage of EPA in their lipids. The fish oil must also be carefully handled and stored during processing to protect against damage which can result in the formation of unacceptable molecular species such as trans EPA which is an unacceptable contaminant in therapeutic formulations and virtually impossible to remove during purification. The complex structure of the fishing industry, the careful handling requirements and the dwindling and finite resource of high EPA fish species means that production of high purity EPA from sea fish is difficult to scale up in order to meet increasing demands and is likely to be unsustainable.
Many publications have reported the potential of alternative sources of EPA-rich compositions or EPA produced from cultured microbes including (micro)algae, fungi, and bacteria. Some of these sources contain low levels of undesired fatty acids. Additionally, the generally less complex fatty acid composition of microbes as compared to fish oil may offer advantages in purification. Variation in fatty acid composition in cultured microorganisms is minimal as compared to fish oil conferring an additional advantage for purification. Production of EPA-rich compositions in biotechnological processes is likely to be rapidly scalable and provide EPA-rich compositions suitable for both nutritional and therapeutic use that are of consistent quality.
The majority of the publications relating to production of EPA from microalgae concern the development of outdoor production systems. The advantage of these systems is the main source of energy for growth,—sunlight, is free. Outdoor production systems however suffer from several key deficiencies. Firstly contamination from competing microorganisms limits the applicability of open pond or raceway cultures to species which are able to withstand environmental conditions that limit the growth of other competing microorganisms. Secondly “photobioreactor” production systems designed to restrict contamination require very large surface to volume ratios to facilitate penetration of light into the culture creating a requirement for large upfront capital expenditure in the establishment of these systems and an ongoing technical challenge and cost with regard to maintaining sterility.
A further weakness of largely photosynthetic cultures developed to date is that species have not yet been isolated that accumulate significant quantities of intracellular lipid in the form of triglycerides when produced photosynthetically. This limits EPA production to that accumulated in polar lipids, the upper limit of which appears to be under tight physiological regulation.
Mixotrophic production systems have been proposed for production of EPA-rich microorganisms. These provide a proportion of the energy for growth in the form of organic carbon supplied to the culture medium. An advantage of mixotrophy includes higher productivities than are achievable with solely photosynthetic production and potentially also lower the overall requirement for light. A disadvantage of the addition of organic carbon sources to outdoor photobioreactor cultures however is the creation of an additional contamination risk by presenting a substrate for growth of non-photosynthetic contaminating organisms.
A number of solely heterotrophic systems for producing EPA-rich microorganisms have been disclosed. These overcome many of the limitations of photosynthetic systems due to their ability to achieve growth of EPA rich species in the absence of light. By eliminating the requirement for light it is possible to significantly reduced the surface-to-volume ratio of reactors and consequently also reduce capital expenditure and sterilisation costs. An additional advantage of heterotrophic production systems is that culture parameters can be tightly controlled leading to production of a product of a consistent quality.
Lipid Classes and Fatty Acid Profile
The fatty acid composition of certain EPA-rich microalgae contain low proportions of fatty acids with structural similarity to EPA. Together with the generally less complex fatty acid composition of microalgae this may offer advantages in terms of purification over fish oil.
In addition to achieving a favorable overall fatty acid composition in cultured EPA-rich microalgae the selective production of EPA in particular lipid classes is also possible.
One particular strategy for enhancement of lipid and overall EPA production in EPA-rich microalgal species is the timed imposition of a nitrogen limitation in microbial culture medium in heterotrophic cultures of microalgae. When microorganisms are deprived of key nutrients required for synthesis of membranes, lipids may be accumulated in the form of triglyceride, a lipid class not utilized extensively in lipid membrane structure.
EPA-rich triglycerides are of potential therapeutic value. EPA may be recovered from triglycerides and further purified via an array of conventional and emerging techniques. Processes designed to extract, concentrate or purify EPA-rich lipid or fatty acid compositions from triglycerides however may be disadvantaged by the presence of a relatively high level and wide range of undesirable fatty acid molecules, and a low level of stereospecificity in terms of the location of EPA within the triglycerides.
Certain polar lipid classes produced in cultures of microalgae are relatively rich in EPA. At the same time some of these lipid classes may exhibit a high degree of stereospecificity in terms of the location of EPA within the class and its isomers. This concentration of EPA in a predictable manner in particular lipid classes provides an additional opportunity to sequester undesirable molecules in unused fractions during a purification process. In addition certain lipid classes produced by cultures of microalgae may also have therapeutic value in their own right.
It may seem surprising then that little, if any, attention has been given to the possibility of inducing heterotrophic or largely heterotrophic cultures of microalgae to localise EPA in polar lipid reservoirs in such a way as to enhance the efficiency and applicability of extraction, concentration and purification processes and to provide a source of polar lipid for incorporation into therapeutic products. In fact prior art disclosures appear to teach away from this possibility.
Unfortunately until now strategies applied to enhancing the productivity of processes providing alternative sources of EPA-rich compositions have led to a reduction in the polar lipid content of EPA-rich microorganisms.
Microalgae produce two major types of polar lipids;—phospholipids and glycolipids. All these major polar lipid classes comprise a glycerol backbone with three positions conventionally labeled Sn 1-3. Phospho and galacto lipid classes are categorised respectively according to phosphate- and galactose-containing functional groups which are attached to the glycerol backbone usually at the Sn-3 position. Fatty acids are acylated at one or more positions 1-2. Isometric forms of these lipid classes arise from acylation patterns where not all available positions are occupied by fatty acids or where a functional group is attached at an alternative position.
Galactolipids are produced predominantly in the chloroplast and are a structural component of the photosynthetic membrane. Galactolipids are one of the most polar of all the lipid classes; there is a substantial difference in charge distribution over the molecule because of the polar nature of the one or more galactose moieties that are attached to the glycerol backbone, providing spatially separated centres of positive and negative charge. Hence galactolipids have found application as emulsifying agents and have been proposed as drug delivery conjugates.
The polar nature (among other physiochemical properties) of galactolipids leads to a number of useful opportunities, including but without limitation to potential advantageous routes for extraction and purification of galactolipids and galactolipid fatty acids, formulation of galactolipids and galactolipid fatty acids into foods, functional foods, beverages, pharmaceutical and industrial compositions, delivery of galactolipids and galactolipid fatty acid nutritional and therapeutic products in a bioavailable form, as well as advantageous therapeutic effects and mechanisms of action their use may promote.
Phospholipids are major structural components of cellular membranes. The highly polar ‘head’ of the molecules coupled with their hydrophobic fatty acid ‘tails’ lead the phospholipids to spontaneously form micelles and bilayers in aqueous media. Phospholipids both within and external to the chloroplast are expected to play a number of important roles in relation to the physiological response of microorganisms to light. For example it has been proposed that fatty acids located in cytoplasmic phospholipids classes are a reservoir for incorporation into chloroplastic lipids during production of photosynthetic membranes. The polar nature of phospholipids among other physiochemical properties presents a number of useful opportunities similar to those stated above for galactolipids. Certain phospholipids including PC are known to be absorbed differentially in mammals which could be turned to a therapeutic advantage. Work on absorption of galactolipids in particular MGDG in mammals is limited.
Prior Art
Culture:
Cohen et al. Journal of Applied Phycology 5: 109-115, 1993 disclose a general scheme for obtaining microalgal galactolipids and producing compositions enriched with the fatty acid GLA. The method disclosed involves extracting the total lipids of the organism and then separating the galactolipids from the total lipid fraction. These authors recognise in the same prior art publication that in order to be industrially useful the content of GLA in the microalgae (and presumably in the galactolipid fraction) would have to be increased. The organisms used in this study were grown under totally photosynthetic conditions. To our knowledge, prior to the present invention neither Cohen and colleagues nor any other previous authors have suggested that the required increases in yield could be accomplished by using largely heterotrophic growth.
Kyle at al in U.S. Pat. No. 5,567,732 disclose a method for producing EPA-rich oils from cells of the diatom Nitzschia alba in the dark and teach that it is possible to induce this organism in heterotrophic culture to enter an oleogenic phase by allowing nitrogen depletion to occur and after 12-24 hours allowing a silicate depletion state to also occur, while continuing to supply other nutrients to the culture. The colourless species of diatoms are preferred. (Colourless species in general and in particular the microorganism preferred by Kyle at al are colourless because they do not exhibit the phenotype of photosynthetic pigments. N. alba for example is believed to be an obligate heterotroph which means that it does not have any active photosynthetic capacity. Nevertheless, a published lipid class analysis of N. alba reports that a few percent of the lipid composition are comprised of galactolipids.) The authors claim that diatoms can successfully be economically cultivated to produce large quantities of single cell oil and they state “for the purposes of this specification, single cell oil means a triglyceride product of a unicellular microorganism”. To our knowledge prior to the present invention neither Kyle et al nor previous authors to our knowledge have disclosed heterotrophic or largely processes useful in the commercial co-production of EPA-rich polar lipids.
The mixotrophic production of the EPA-rich microalga Phaeodactylum tricornutum in a tubular photobioreactor is disclosed in Ceron Garcia et al Journal of Applied Phycology 12: 239-248, 2000. This manufacture utilizes 9.2 g L−1 glycerol as an organic carbon source and supplies an external irradiance of 165 μmol photons m−2 s−1 to the photobioreactor surface. These authors claim that by reducing the need for light this form of mixotrophic growth has a number of advantages including the possibility of greatly increasing the algal cell concentration and EPA productivity in outdoor mass culture on a large-scale. Ceron Garcia and colleagues did not however identify the advantages of largely heterotrophic growth in terms of increased polar lipid production. Furthermore neither these authors nor any previous authors have identified 290 the potential for utilizing relatively low levels of irradiance in largely heterotrophic culture for producing EPA-rich microorganisms.
A number of prior art publications have disclosed that culture conditions including light intensity and wavelength can enhance lipid and overall EPA production in specified EPA-rich microalgal species. However, prior to the present invention it had not been proposed that sub photosynthetic light intensities could be used to alter the relative production of lipid classes in a commercially useful manner. Nor had it been proposed that sub photosynthetic light intensities could be utilized to alter the localization of EPA in lipid reservoirs of microalgae in a commercially useful manufacture.
Extraction of Polar Lipids from Microalgae
Several techniques of potential industrial utility have been proposed for extraction and concentration of galactolipids and/or fatty acids from galactolipid fractions of biological material. Winget (U.S. Pat. No. 5,767,095) describes in detail a range of extraction and concentration techniques used to recover particular lipid classes, including relatively pure galactolipids containing EPA, from a number of photosynthetically produced microalga including those of the diatom genus Chlorella. 
Cohen et al (J. Appl. Phycol. 5: 109., 1993.) disclose the fatty acid DHA may be produced from the phosphatidylethanolamine (PE) lipid fraction of the photosynthetic organism Isochlysis galbana by extracting total lipids and subsequently producing DHA rich compositions by employing the well known technique of urea crystallisation. Vali et al U.S. Pat. No. 6,953,849 disclose a process involving dewaxing of rice bran and hexane extraction and includes HPLC with a silicic acid column. Colarow U.S. Pat. No. 5,284,941 discloses a method involving solvent boric acid gel separation. Buchholz et al U.S. Pat. No. 5,440,028 discloses a method isolation through membrane separation, with pH adjustment. Bergqvist et al 1995 report after their work on oat kernels that galactolipids may be commercially extracted from a range of biological materials using a solid phase extraction using the known differences in solubility in acetone between phospholipids and glycolipids. They started with a hot ethanol extraction then used hexane then hexane/acetone then acetone.
No prior art publications are known to teach that it is possible to selectively isolate EPA-rich compositions in a commercial manufacture from the lipids of organisms that have been cultured using largely heterotrophic culture capable of enhancing EPA productivity and simultaneously increasing the concentration of EPA in specific polar lipid fractions.
Enzymatic Purification
A number of prior art publications disclose the use of enzymes to liberate lipids and arrive at concentrated and purified lipid and fatty acid containing compositions from fish oil and other starting materials. The inventors appreciate that various lipases and phospholipases are capable of dis-assembling lipids. For example a variety of solvent-based extraction systems and crystallisation techniques have been disclosed that favour extraction of lipids of a particular class or fatty acids of a particular chain length or degree of unsaturation. These enzymes which may include lipases and proteases are known to act preferentially on different substrates. In the case of lipases for example, enzymes are expected to some degree to be specific for lipid class, fatty acid, and the position of the fatty acid within the lipid class. The activity and preference of enzymes can be altered by altering environmental conditions such as temperature, and via the addition of cofactors and techniques such as immobilization.
A common analytical technique used to estimate the localisation of fatty acids at different positions in the lipid structure is to expose a fatty acid class to a lipase capable of selectively hydrolysing fatty acids located in a particular position. It follows that the common general knowledge of those skilled in the art includes the recognition that both the proportion of lipids and the localisation of target fatty acids as well as co-localisation or lack thereof of undesired acids within lipid reservoirs of a biological material constitute critical aspects in a purification process at an analytical scale. To our knowledge however no previous authors have disclosed methods of producing therapeutic or prophylactic compositions via the selective enzymatic hydrolysis of algal polar lipids at least not from polar lipids produced in largely heterotrophic cultures.
Applications of Galactolipids
Winget teaches use of topically applied MGDG-EPA compositions in the prevention and treatment of inflammation, but does not disclose application of lipase-type or indeed any enzymes. Later, Bruno et al (Eur J Pharmacol: 524; 159-168 7 Nov. 2005) disclose that the galactolipid classes MGDG, DGDG and SQDG obtained from thermophilic blue-green algae have in-vivo anti-inflammatory activities in a croton-oil induced mouse ear inflammatory response. However there is no indication in the abstract that any of the n-3 HUFAs were present.